Transcription of the HIV genome during virus replication shows distinct kinetic phases (see references 53, 59, 60, 79). The initial products of HIV gene expression are short, multiply spliced mRNAs approximately 1.8 to 2.0 kb in length, which encode the trans-acting regulatory proteins tat, rev (and possibly nef). As infection by the virus develops, and the levels of the tat and rev proteins rise in the infected cells, mRNA production shifts progressively towards production of a family of singly-spliced 4.3 kb mRNAs encoding env and other HIV gene products such as vif and vpr. Finally, late in the infection process, production switches to fill-length, unspliced, transcripts which act both as the virion RNA and the mRNA for the gag-pol polyprotein.
To achieve this control of gene expression, the HIV virus relies on the interaction of cellular and virus-encoded trans-acting factors with cis-acting viral regulatory sequences (1, 3, 53). Initiation of transcription relies largely on the presence of binding sites for cellular transcription factors in the viral long terminal repeat (LTR) (28). In contrast, the virally encoded regulatory proteins tat and rev exert their activity via cis-acting sequences encoded within HIV messenger RNAs. The transactivation-responsive region (TAR) is required for tat activity, and is located in the viral long terminal repeat (LTR) between residues +1 and +79 (5, 9, 10, 11, 12, 13, 14, 16, 27, 38). The rev-responsive element (RRE) has been localized to a 243-nucleotide long sequence within the env gene (47, 51, 54, 65, 67, 68, 77). Similar regulatory proteins and target sequences are used by HIV-2 and SIV (8, 66). The HTLV-1 virus rex gene product appears to function analogously to rev, and can functionally substitute for rev to promote viral gene expression (76).
The distinct kinetic phases of HIV transcription are now believed to reflect the intracellular levels of the regulatory proteins tat and rev. Initially, binding of host transcription factors to the LTR induces basal level transcription of the early mRNAs including tat. As tat levels rise, increased transcription from the LTR is stimulated by the trans-activation mechanism. This leads to further increases in tat levels, and also stimulates production of rev. Production of the viral structural proteins begins once rev levels have risen to sufficiently high levels to promote export of messenger RNAs carrying the rev-responsive element (RRE) sequence. The HIV growth cycle may also include a latent stage where viral gene expression is silent because transcription from the viral LTR produces insufficient amounts of regulatory proteins to initiate the lytic growth cycle. Significant levels of HIV gene expression are only achieved in the presence of tat protein. Initially, it seemed most likely that tat activity produced an increased rate of transcription initiation either by stimulating binding of host transcription factors to sequences in viral LTR or by acting as a transcription factor itself (1, 3, 53). However, several experiments strongly suggest that tat activity requires RNA target sequences rather than DNA target sequences.
Deletion analysis of the viral LTR showed that tat activity requires a regulatory element located downstream of the initiation site for transcription, at the 5-terminus of all the mRNA transcripts between residues +1 and +79, called the trans-activation-response region (TAR) (5, 10, 11, 27, 28). The placement of TAR in a transcribed region was surprising, since this suggested that it could function as an RNA rather than as a DNA element. Support for this idea came from observation that unlike enhancer elements, the TAR element is only functional when it is placed 3' to the HIV promoter, and in the correct orientation and position (5, 12-15). Furthermore, the TAR RNA sequence forms a highly stable, nuclease-resistant, stem-loop structure, and point mutations which destabilize the TAR stem by disrupting base-pairing usually abolish tat-stimulated transcription (88).
Berkhout et al. (16) provided convincing evidence that the TAR RNA sequence must be transcribed in the nucleus and correctly folded in order for trans-activation to occur. They introduced antisense sequences, designed to destabilize formation of TAR RNA hairpin-loop structures either upstream or downstream of TAR. Both the correct folding of TAR and trans-activation were blocked when the destabilizing sequences were placed on the 5'-side of TAR, but placement of the same sequences on the 3'-side of TAR allowed normal trans-activation. This strongly suggested that the TAR hairpin-loop structure could fold and become active on nascent transcripts.
Trans-activation does not appear to be due to tat regulation of DNA-binding proteins to upstream promoter elements (17) since viral LTRs truncated upstream of the Spl sites (18) or fused to heterologous promoters (5, 19, 20) may be trans-activated.
In tat-expressing cells both mRNA levels, as measured by hybridization, and nuclear transcription rates, as measured by run-on experiments, are increased 7 to 40 fold (88, 89). Although there has been considerable debate about whether tat could also increase the translation efficiency of mRNAs carrying TAR, it now seems clear that the increase in mRNA synthesis promoted by tat is sufficient to account for trans-activation. Accurate measurements of both CAT mRNA and CAT protein levels in transfected cells, show that protein accumulated in parallel to the mRNA over a broad range. At very low levels of CAT mRNA translation is less efficient than at high levels of message. However this increase in RNA utilization is not tat-specific since any increase in mRNA levels, such as after stimulation of transcription by adenovirus EIA protein, produces a corresponding effect on translation (89).
One case where translational control of TAR-containing mRNAs by tat has been reported to be active is in Xenopus oocytes. When CAT mRNAs carrying TAR sequences are injected into the cytoplasm of Xenopus oocytes they are not translated, but translation is restored after co-injection of the mRNA into the nucleus together with tat protein (90). This phenomenon could be a reflection of a specialized mechanism in Xenopus; translational control by tat could not be demonstrated when parallel microinjection experiments were performed in mammalian cells permissive for HIV growth (91). It is also possible that the translation block observed in the Xenopus experiments is due to contamination of the mRNA preparations by double-stranded RNA fragments produced during in vitro transcription by bacterial RNA polymerases. In mammalian cell free translation systems TAR-containing mRNAs were reported to be translated poorly because the TAR sequence appeared to activate the double-stranded RNA-dependent kinase (92). However, after re-purification of preparations of the TAR-containing-RNAs by chromatography on cellulose columns, contaminating double-stranded RNAs were removed and the TAR-containing mRNA preparations were translated normally (93).
Although the simplest explanation for the ability of tat to stimulate transcription from promoters that carry the TAR sequence is that tat recognizes TAR RNA by direct binding, demonstration of this interaction proved to be difficult. Early attempts to demonstrate specific binding of tat to TAR RNA were probably unsuccessful because it is difficult to purify tat expressed in E. coli free from RNA contaminants and under conditions where aggregates are not formed due to oxidation of its seven cysteine residues. However, using an improved method we were able to demonstrate that tat is able to specifically recognize TAR RNA. Binding shows high affinity (K.sub.d =12 nM) and tat forms one-to-one complexes with TAR RNA (94).
Tat recognition of TAR requires only the presence of a U-rich bulge near the apex of the TAR RNA stem as well as closely flanking base-pairs. Mutations which alter the U-rich bulge sequence, or which affect the structure of the U-rich bulge by disrupting base-pairing in nearby residues of the TAR stem-loop structure, abolish both tat binding and trans-activation (10, 11, 27, 28). By contrast, the identity of many of the base pairs throughout the TAR stem does not appear to be an important requirement for transactivation or for tat binding so long as Watson-Crick base pairing is maintained. RNA binding by tat almost certainly involves the formation of specific salt bridges between arginine residues on tat and phosphates on the TAR RNA. C-terminal peptides carrying an arginine-rich sequence from tat are also able to bind to TAR RNA at the U-rich bulge, although the peptides bind with less specificity and lower affinity than the intact protein (95, 96).
Experiments using hybrid proteins containing tat and an exogenous RNA-binding domain have provided interesting genetical evidence supporting the biochemical evidence that tat is presented to the transcription machinery after binding directly to nascent transcripts carrying the TAR RNA element (97, 98). For example, fusion proteins containing sequences from tat and bacteriophage R17 coat protein can stimulate transcription from HIV LTRs when the TAR RNA sequence is replaced entirely by a RNA stem-loop structure carrying the RI 7 operator sequence. As in the case of tat recognition of TAR RNA, binding appears to be direct, since mutations in the RI 7 RNA operator sequence that reduce its affinity for coat protein produce a corresponding decrease in trans-activation by the tat RI 7 fusion protein in vivo (98).
How does tat regulate gene expression after binding to TAR RNA? One early suggestion was that TAR acts to stimulate transcription initiation rates, by acting as an additional loading site for transcription factors (99). Although difficult to rule out, this model seems increasingly unlikely since hybrid promoters including promoters that contain only binding sites for fusion proteins containing the yeast GAL-4 binding domain are highly responsive to tat. It should also be noted that there are no known examples of RNA binding proteins that affect transcriptional initiation whereas there are many good examples in prokaryotic systems of RNA binding proteins, such as the bateriophage lambda N protein, that control transcriptional elongation.
Peterlin and his colleagues made the important suggestion that tat acts as an anti-terminator which helps to overcome a block to elongation at or near the TAR site (38, 40, 98). Their proposal is based on observations that short, prematurely-terminated RNA transcripts accumulate in the absence of tat. However TAR is not simply a site of anti-termination since mutations in TAR which abolish trans-activation, or deletions of TAR, do not result in constitutively high levels of LTR expression and it is difficult to "chase" the short RNA transcripts into full-length mRNAs. The failure to identify a specific terminator sequence downstream of TAR has led to a third proposal in which tat acts as a more general elongation factor for RNA polymerase II and not simply as a site-specific anti-termination factor. It seems likely that tat and cellular co-factors assemble with the RNA polymerase II soon after transcriptional initiation in a reaction mediated by the protein binding sites present on the TAR-containing nascent transcripts. This modified transcription complex then stimulates viral mRNA production by overcoming additional blocks to elongation at a variety of distal sites, including TAR.
Strong support for the elongation factor model comes from nuclear run-on experiments. In the absence of tat RNA polymerases can only be found at or near the viral LTR. However, the density of RNA polymerases downstream of the promoter increases dramatically in the presence of tat (89). However, a rigorous demonstration of an elongation-dependent mechanism will require the development of efficient cell free transcription systems that respond to tat. There are encouraging signs that these will soon be available. Addition of extracts from HIV-infected cells stimulates transcription from the viral LTRs (100). There has also been a recent report that bacterially synthesized tat can stimulate transcription in vitro. Unfortunately, stimulation of transcription by the recombinant tat is best observed only after extended incubations with high concentrations of added protein (101). It therefore remains unclear whether genuine trans-activation has been reproduced in vitro, and additional development of the cell free systems will be required before mechanistic studies can begin.
It is an aim of the present invention to provide an effective method for, and compositions for use in, the inhibition of HIV viral growth within cells, which involves modifying the activity of the regulatory protein tat in the viral growth cycle, and also an assay for screening potential anti-viral agents.